CN110357896A - A kind of compound and preparation and its application in detection bivalent cupric ion and strong acid pH - Google Patents

Abstract

The present invention relates to a class of fluorescent sensors in the field of fine chemicals, and their preparation methods and uses, in particular to a class of dual-function recognition sensors based on rhodamine dyes and long-wavelength Cy7 Qing dyes and their preparation methods, which can detect divalent copper ions by near-infrared ratios. And the application of pH and fluorescence microscopy imaging detection of divalent copper ions in cells. The copper ion fluorescence sensor based on ethylenediamine-bridged rhodamine B and Cy7 cyanine dyes of the present invention has the following remarkable features: (1) buffered in acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) In solution, near-infrared ratiometric detection of divalent copper ions can be achieved; (2) good selectivity, almost no response to other metal ions; (3) detection of divalent copper ions is not sensitive to pH; (4) near-infrared ratiometric Detect the pH value of strong acid system; (5) The selectivity of detecting pH is good.

Description

A class of compounds and their preparation and their application in the detection of divalent copper ions and strong acid pH

technical field

The present invention relates to a class of fluorescent sensors in the field of fine chemicals, and their preparation methods and uses, in particular to a class of dual-function recognition sensors based on rhodamine dyes and long-wavelength Cy7 Qing dyes and their preparation methods, which can detect divalent copper ions by near-infrared ratios. And strong acid pH and the application of fluorescence microscopy imaging of divalent copper ions in cells and bacteria under strong acid pH.

Background technique

Copper exists in all living organisms and is an important trace element in redox, growth and development processes. The content of copper in cells ranks only after iron and zinc. Copper plays an important role as a catalytic cofactor in different cellular physiological processes, such as mitochondrial respiration, iron uptake, and redox processes of a large number of enzymes, including Cytochrome oxidase, superoxide dismutase, ascorbate oxidase and tyrosinase, etc. Copper requires a tightly controlled balance in living organisms to ensure an adequate supply without toxic effects. Excess or deficiency of copper has been associated with Wilson's disease, Menkes syndrome, ALS disease and Alzheimer's disease, respectively. Therefore, the detection of Cu ²⁺ concentration is of great significance. There are different test methods available to determine the amount of copper in a sample. However, many methods have the disadvantages of only determining the total content of copper in a given sample, destroying the sample, and expensive instruments. Therefore, these methods are not suitable for the determination of intracellular copper ion content and distribution, so their application is limited. In contrast, the fluorescent probe method has been widely valued due to its special photophysical and photochemical properties, which have the advantages of simple operation, high sensitivity, low detection limit, and non-invasiveness. In particular, near-infrared bioluminescent imaging has high resolution, can penetrate thick tissues, has little damage to biological samples, and is less interfered by background autofluorescence of biomolecules. At the same time, ratiometric fluorescence recognition of Cu ²⁺ can be automatically calibrated to eliminate certain factors. Interference improves the accuracy of Cu ²⁺ identification.

pH value is a very important parameter in many fields of chemical reaction, biology, medicine and industrial production. Accurate measurement of pH is of great significance. Abnormal pH values in cells and tissues are often associated with diseases such as cancer and Alzheimer's. Therefore, developing methods to monitor pH changes in living cells and tissues is an important goal in the field of life sciences. Fluorescence spectroscopy for pH measurement has attracted extensive attention due to its advantages of high sensitivity, fast response, convenient operation, non-invasive detection, and continuous monitoring of rapid and dynamic changes in pH.

In view of the good detection technology provided by the near-infrared ratiometric fluorescence detection method, it has been widely used in the analysis and detection of heavy metal ions such as Cu ²⁺ , Zn ²⁺ , Hg ²⁺ and pH. Fluorescent probes based on dyes such as rhodamine, phenanthrene, fluorescein, and cyanine dyes have been reported in the literature for dual recognition detection of Cu ²⁺ and pH. However, it is still a challenge to find fluorescent sensors with good fluorescence properties (such as high quantum yield, long wavelength and stable properties) that can detect Cu2 ⁺ and strong acid pH in real time. We synthesized a class of rhodamine B and Cy7 cyanine dyes bridged with ethylenediamine for copper ions and strong acid pH fluorescent sensors, which can achieve near-infrared ratiometric fluorescence selectivity for Cu ²⁺ and proton OFF-ON in aqueous solution rapidly. response. This type of fluorescent sensor can be used for real-time on-line detection of divalent copper ions in living cells and pH detection of Escherichia coli in a strong acid system, and therefore has good economic effects.

Contents of the invention

The present invention aims to provide a class of copper ion fluorescence sensors based on ethylenediamine bridged rhodamine B and Cy7 cyanine dyes, which can be used for the quantitative detection of protons and Cu ²⁺ in water samples, as well as the detection of the content and concentration of Cu ²⁺ in living cells. Distribution, and pH detection of bacteria in strong acid systems.

The present invention is achieved through the following technical solutions: a class of compounds has the following general structural formula I:

In general formula I: a is 1-6.

The present invention further provides a preparation method for the above-mentioned class of compounds, obtained by reacting compound II with general structural formula III in N,N-dimethylformamide solution,

The present invention further provides a preparation method of one of the compounds, dissolving 2,3,3-trimethyl-3H-indoline in acetonitrile and reacting with 6-bromohexanoic acid to generate compound IV, compound IV and condensed Compound 2-chloro-1-formyl-3-hydroxymethylcyclohexene is reacted to generate compound III; rhodamine B is dissolved in ethanol and reacted with ethylenediamine to generate compound II; finally, compound III and compound II are dissolved in N , Compound I is obtained after reacting in N-dimethylformamide;

The compound IV is:

The compound III is:

The compound II is:

The compound I is:

As a further improvement of the technical scheme of the compound I preparation method, the molar ratio of 2,3,3-trimethyl-3H-indoline to 6-bromohexanoic acid is 1:1-3.

As a further improvement of the technical scheme of the preparation method of compound I, the molar ratio of the condensing agent 2-chloro-1-formyl-3-hydroxymethylcyclohexene to compound IV is 1:2.1-5.

As a further improvement of the technical scheme of the compound I preparation method, the molar ratio of rhodamine B to ethylenediamine is 1:1-5.

As a further improvement of the technical scheme of the compound I preparation method, the molar ratio of compound III to compound II is 1:1-5.

The present invention further provides the application of this type of compound as a ratio-type fluorescent sensor in detecting pH value. And the application of this type of compound as a ratiometric fluorescent sensor in the detection of divalent copper ions.

The synthesis and analytical detection methods of the ratiometric fluorescent sensor RCy7 are described in more detail in the examples of this specification.

Fluorescence sensor RCy7 of the present invention has very good selectivity to Cu ²⁺ in acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) buffer solution, before adding Cu ²⁺ , use Excited at 550nm, the fluorescence signal at 722nm (Cy7 Qing dye) was collected, and the fluorescence intensity ratio (F ₅₇₇ /F ₇₂₂ ) was 0.44. With the addition of Cu ²⁺ , the fluorescence at 722nm weakened, while the fluorescence signal of rhodamine (577nm ) gradually increased. When 5 equivalents of Cu ²⁺ were added, the fluorescence intensity ratio (F ₅₇₇ /F ₇₂₂ ) was 26.25, which was increased by about 59.66 times. The shift of the two emission peaks exceeds 145nm, which realizes the effective discrimination of emission doublets in the ratiometric fluorescent probe, and realizes the near-infrared ratiometric fluorescence detection of Cu ²⁺ in vitro and in cells.

Fluorescence sensor RCy7 of the present invention also has very good selectivity to proton in acetonitrile/water buffer solution, when pH=6.58, use 550nm to excite, collect the fluorescent signal of 722nm (Cy7 blue dye), fluorescence intensity The ratio (F ₅₈₀ /F ₇₂₂ ) is 0.35. As the proton concentration increases, the fluorescence at 722nm weakens, while the fluorescence signal (580nm) of rhodamine increases gradually. When pH=1.26, the fluorescence intensity ratio (F ₅₈₀ /F ₇₂₂ ) is 167.33, an increase of about 478.09 times. The shift of the two emission peaks exceeds 141nm, realizing the ratiometric fluorescence detection of pH in vitro. At the same time, pH near-infrared fluorescence microscopy imaging of E.coli bacteria under different pH conditions was also realized.

The fluorescence sensor RCy7 realizes ratiometric detection of near-infrared fluorescence for divalent copper ions with a small detection limit; it can also realize ratiometric detection of near-infrared fluorescence for pH. The fluorescence intensity ratio (F ₅₇₇ /F ₇₂₂ ) has a good linear relationship with the concentration of Cu ²⁺ . In view of this, it can be applied to the detection of divalent copper ions in acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) buffer solution and the fluorescence microscopy imaging of copper ions in biological living cells, as well as in environmental water The detection of divalent copper ions and protons in the system can also realize near-infrared fluorescence microscopy imaging of strong acid pH of E.coli bacteria. Therefore, it has a good application prospect.

The copper ion fluorescent sensor based on ethylenediamine-bridged rhodamine B and Cy7 cyanine dyes of the present invention has the following remarkable features:

(1) In the acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) buffer solution, the near-infrared ratiometric detection of divalent copper ions can be realized;

(2) Good selectivity, almost no response to other metal ions;

(3) Detection of divalent copper ions is not sensitive to pH;

(4) Near-infrared fluorescence microscopy imaging of copper ions in living cells can be realized;

(5) The pH value of the strong acid system can be detected by near-infrared ratio;

(6) The selectivity of detecting pH is good;

(7) The strong acid pH near-infrared fluorescence microscopy imaging of E.coli bacteria can be realized.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. Those skilled in the art can also obtain other drawings based on these drawings without creative work.

Fig. 1 is the proton nuclear magnetic resonance spectrogram of the present invention to compound III in CDCl ₃ reagent.

Fig. 2 is the carbon nuclear magnetic resonance spectrum of the present invention to compound III in CDCl ₃ reagent.

Fig. 3 is the high-resolution mass spectrum of compound III according to the present invention.

Fig. 4 is the hydrogen nuclear magnetic resonance spectrum of the fluorescent sensor RCy7 of the present invention in CDCl ₃ reagent.

Fig. 5 is the carbon nuclear magnetic resonance spectrum of the fluorescent sensor RCy7 of the present invention in CDCl ₃ reagent.

Fig. 6 is a mass spectrum of the fluorescence sensor RCy7 of the present invention.

Fig. 7 is a graph showing the relationship between the absorbance and the concentration of divalent copper ions of the fluorescence sensor RCy7 (10 μmol/L) of the present invention in acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) solution. The abscissa is the wavelength (nm), and the ordinate is the absorbance. The concentration of the fluorescent sensor RCy7 is 10 μmol/L, and the arrow indicates that the concentration of copper ions varies from small to large in order of 0, 1, 3, 5, 7, 10, 15, 20, 30, 50, 50, 70, 100, 130, 150, 180, 200, 250, and 300 μmol/L.

Fig. 8 is a graph showing the relationship between fluorescence intensity and divalent copper ion concentration of the fluorescence sensor RCy7 (10 μmol/L) of the present invention in acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) solution. The abscissa is the wavelength (nm), and the ordinate is the fluorescence intensity. The concentration of the fluorescent sensor RCy7 is 10 μmol/L, and the arrow indicates that the concentration of divalent copper ions changes from small to large in order of 0, 1, 3, 5, 7, 10, 12, 13, 14, 15, 20, 30, 35, 40, 45, 50, 70, 100, 130, 150, 180, 200, 250, 300 μmol/L. Excitation wavelength: 550nm.

Figure 9 is the ultraviolet-visible spectrum of the fluorescent sensor RCy7 (10 μmol/L) of the present invention after adding different metal cations (50 μmol/L) to the acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) solution picture. The abscissa is the wavelength (nm), and the ordinate is the absorbance.

Fig. 10 is the fluorescence spectrum of the fluorescence sensor RCy7 (10 μmol/L) of the present invention after adding different metal cations (50 μmol/L) to the acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) solution. The abscissa is the wavelength (nm), and the ordinate is the fluorescence intensity. Excitation wavelength: 550nm.

Fig. 11 is the fluorescent sensor RCy7 (10 μmol/L) of the present invention, after adding different metal cations (50 μmol/L) into the acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) solution, and then adding 50 μmol/L Cu ²⁺ fluorescence response map of L. The abscissa indicates different metal ions and the probe RCy7, and the ordinate is the fluorescence intensity ratio (F ₅₇₇ /F ₇₂₂ ). Excitation wavelength: 550nm.

Fig. 12 is the fluorescence sensor RCy7 (10 μmol/L) of the present invention after adding different metal cations (50 μmol/L) in acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) solution, under natural light photo.

Fig. 13 is a fluorescence spectrum diagram of the fluorescence sensor RCy7 (10 μmol/L) in acetonitrile/water solution at different pHs of the present invention. The abscissa is the wavelength (nm), and the ordinate is the fluorescence intensity. Excitation wavelength: 550nm.

Fig. 14 is the fluorescence spectrum diagram of the fluorescence sensor RCy7 (10 μmol/L) of the present invention after adding different metal cations (50 μmol/L) in acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) solution, There is also a fluorescence spectrum diagram of the fluorescence sensor RCy7 (10 μmol/L) of the present invention at pH=1.26. The abscissa is the wavelength (nm), and the ordinate is the fluorescence intensity. Excitation wavelength: 550nm.

Fig. 15 is a laser confocal fluorescence microscopy image of the fluorescence sensor RCy7 of the present invention in Hela cells. (A) Hela cells were cultured with fluorescent sensor RCy7 (10 μmol/L) (ae), and (B) 50 μM Cu ²⁺ was added to Hela cells cultured with fluorescent sensor RCy7 (10 μmol/L) (fj). Among them, a, f are the fluorescence images in the 560-650nm channel; b, g are the fluorescence images in the 680-800nm channel; c, h are the bright field images of a and f respectively; d is the overlay image of a and c; e is b and c; i is the overlay of f and h; j is the overlay of g and h. Excitation wavelength in the figure: 552 nm, scale bar: 25 μm.

Fig. 16 is a laser confocal fluorescence microscopy image of the fluorescence sensor RCy7 (10 μmol/L) of the present invention cultured at pH (1.8) and pH (7.4) of Escherichiacoli bacteria. (A) cultured Escherichia coli bacteria (a-e) with fluorescent sensor RCy7 (10 μmol/L) at pH = 7.4, (B) cultured Escherichia coli bacteria with fluorescent sensor RCy7 (10 μmol/L) at pH = 1.8 (f-j). Among them, a, f are the fluorescence images in the 560-650nm channel; b, g are the fluorescence images in the 680-800nm channel; c, h are the bright field images of a and f respectively; d is the overlay image of a and c; e is b and c; i is the overlay of f and h; j is the overlay of g and h. Excitation wavelength in the figure: 552 nm, scale bar: 25 μm.

Detailed ways

In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Apparently, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other implementations obtained by persons of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

The technical solution of the present invention will be described in detail below in conjunction with the accompanying drawings. Those skilled in the art should also recognize that: the characteristics of the individual compounds (a in the general structural formula I is 5) demonstrated in the examples of the present invention, other non-exemplified compounds of the present invention also have the same, and they are also used in the detection of bivalent compounds. Copper ions have a similar effect in strong acid pH.

A class of compounds described in the present invention has the following general structural formula I:

In general formula (I): a is 1-6.

In a specific embodiment, said a is preferably 1-6, most preferably 5.

An example of preferred technical features is one of the specific embodiments of the present invention. The fluorescent sensor described in the present invention is selected from the following compounds:

The reaction scheme of compound RCy7 shown above is as follows:

On the other hand, the present invention provides the preparation method of the above-mentioned fluorescent sensor, which is to dissolve 2,3,3-trimethyl-3H-indoline in acetonitrile and react with 6-bromohexanoic acid to generate compound IV, compound IV and The condensing agent 2-chloro-1-formyl-3-hydroxymethylcyclohexene reacts to generate compound III. Rhodamine B was dissolved in ethanol and reacted with ethylenediamine to generate compound II. Finally, compound III and compound II were dissolved in N,N-dimethylformamide and reacted to obtain fluorescent sensor I (RCy7).

In a specific embodiment, the molar ratio of the 2,3,3-trimethyl-3H-indoline to 6-bromohexanoic acid is 1:1-3, preferably 1:1.2-2, the best is 1:1.5, a substitution reaction occurs to generate a compound with the general structural formula Ⅳ. This reaction can be carried out in an organic solvent. Organic solvents include but not limited to acetonitrile, ethanol, etc., preferably the reaction solvent is acetonitrile. The reaction temperature is 65°C-81.6°C, preferably 80°C-81.6°C. The reaction process was judged by thin-layer chromatography (TLC) to determine the end point of the reaction.

In a specific embodiment, the molar ratio of the condensing agent 2-chloro-1-formyl-3-hydroxymethylcyclohexene to the compound IV is 1:2.1-5, preferably 1:2.2-3, the best The ratio is 1:2.5, and the reaction takes place to generate a compound with the general structural formula III. This reaction can be carried out in an organic solvent. The organic solvent must include n-butanol. Other organic solvents include but not limited to tetrahydrofuran, 1,4-dioxane, toluene, etc. The preferred reaction solvent is toluene. The reaction temperature is 100°C-115°C, preferably 110°C-115°C. The reaction process was judged by thin-layer chromatography (TLC) to determine the end point of the reaction.

In a specific embodiment, the molar ratio of rhodamine B to ethylenediamine is 1:1-5, preferably 1:1-3, and most preferably 1:1.5, and the reaction generates a compound with a general structure of II . This reaction can be carried out in an organic solvent. Organic solvents include but are not limited to methanol, ethanol, etc., and the preferred reaction solvent is ethanol. The reaction temperature is 60°C-78°C, preferably 78°C. The reaction process was judged by thin-layer chromatography (TLC) to determine the end point of the reaction.

In a specific embodiment, the molar ratio of the compound III to the compound II is 1:1-5, preferably 1:1.5-3, most preferably 1:1.5, and react to form the target compound with the general structural formula I. This reaction can be carried out in an organic solvent. The organic solvent includes but not limited to N,N-dimethylformamide (DMF), methanol, ethanol, etc., and the preferred reaction solvent is DMF. The base to be added in the reaction includes but not limited to triethylamine, diisopropylamine, preferably triethylamine. The reaction temperature is 25°C-80°C, preferably 65°C. The reaction process was judged by thin-layer chromatography (TLC) to determine the end point of the reaction.

The separation and purification method of the fluorescence sensor of the present invention adopts a conventional method and is not particularly limited. Generally, after the reaction is completed, the product is separated and purified by chromatographic column after filtration, evaporation of the solvent, and drying.

The resulting fluorescent sensor can be recovered by separation and purification techniques known in the art to achieve the desired purity.

Various reagents and raw materials used in the present invention are commercially available. Alternatively, it can be simply prepared from raw materials known in the art by methods known to those skilled in the art or methods disclosed in the prior art.

The fluorescent sensor described herein can implement quantitative detection of divalent copper ions in acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) buffer solution; it can detect divalent copper ions in living Hela cells Fluorescence microscopy imaging of Escherichia coli bacteria in strong acid system.

In order to make the present invention more fully understood by those skilled in the art, the following will be described in detail with reference to the drawings and embodiments. These examples are illustrative only and do not limit the invention in any way.

Example 1: Preparation of fluorescent sensor RCy7

Step 1: Synthesis of Compound IV

Dissolve 10 g of 2,3,3-trimethyl-3H-indoline in acetonitrile, add 1.5 equivalents (moles) of 6-bromohexanoic acid dropwise at room temperature, reflux and stir for 20 hours under nitrogen protection, cool, and add diethyl ether Extract, evaporate the solvent, dissolve in a small amount of ethanol, add ether to precipitate, filter, and dry in vacuo. Compound IV (1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indole) was obtained as a white solid.

Step 2: Synthesis of Compound III

Take 1g of condensing agent 2-chloro-1-formyl-3-hydroxymethylcyclohexene, 2.5 equivalents (moles) of compound IV, 50mL of n-butanol, and 5mL of toluene into a flask equipped with a water separator. The mixture was stirred under reflux for 15 hours under the protection of nitrogen to obtain a dark green solution, and the solvent was evaporated by rotary evaporation. Then install a short column and wash with n-hexane/ethyl acetate and dichloromethane/methanol to obtain the crude product. Use gradient n-hexane/ethyl acetate, methylene chloride/methanol solvent system to separate on silica gel chromatography column, obtain dark green solid product compound III (1-(6-butoxy-6-oxohexyl)-2-( (E)-2-((E)-3-((E)-2-(1-(6-butoxy-6-oxohexyl)-3,3-dimethylindole-2-ylidene (yl)ethylidene)-2-chlorocyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-3H-indole).

Step 3: Synthesis of Compound II

Add 10 g of Rhodamine B into 100 mL of ethanol, and then add 1.5 equivalents (moles) of ethylenediamine, and stir and reflux for two hours under nitrogen protection. After cooling, the solvent was evaporated, and the initial product was subjected to silica gel chromatography (the eluent was petroleum ether (60-90°C)/ethyl acetate) to separate and obtain a white solid compound II (2-(2-aminoethyl)-3', 6'-bis(diethylamino)spiro[isoindole-1,9'-xanthene]-3-one).

Step 4: Synthesis of sensor RCy7

Add 2 g of compound III to 30 mL of DMF, then add 1.5 equivalents (moles) of compound II, add 1 mL of triethylamine, and stir at 65°C for 8 hours under nitrogen protection. Cooling, solvent removal under reduced pressure, silica gel chromatography (eluent is dichloromethane/methanol) separation to obtain the blue fluorescent sensor RCy7 (2-((E)-2-((E)-2-( (2-(3',6'-bis(diethylamino)-3-oxaspiro[isoindole-1,9'-oxanthene]-2-yl)ethyl)amino)-3-( (E)-2-(1-(6-butoxy-6-oxohexyl)-3,3-dimethylindol-2-ylidene)ethylene)cyclohexyl-1-ene-1 -yl)vinyl)-1-(6-butoxy-6-oxohexyl)-3,3-dimethyl-3H-indole).

In order to show that the structure of the fluorescent sensor of the present invention is correct, the present invention has done the following test analysis to compound (Ⅲ) and sensor RCy7:

Compound (Ⅲ) proton nuclear magnetic resonance signal in deuterated chloroform is (500MHz, chemical shift, unit ppm): ¹ HNMR (500MHz, CDCl ₃ ) δ8.33(d, J=14.0Hz, 2H), 7.38(d ,J=7.4Hz,4H),7.25(d,J=7.4Hz,2H),7.18(d,J=7.4Hz,2H),6.25(d,J=14.0Hz,2H),4.23(t,J =6.9Hz,4H),4.03(t,J=6.9Hz,4H),2.73(s,4H),2.32(t,J=7.4Hz,4H),2.08(s,1H),1.99(d,J =5.2Hz,2H),1.92–1.81(m,4H),1.72(d,J=7.7Hz,15H),1.61–1.49(m,8H),1.34(dd,J=14.0,7.4Hz,4H) , 0.91 (t, J=7.4Hz, 6H). See Figure 1.

The C NMR signal of compound (Ⅲ) in deuterated chloroform is (125MHz, chemical shift, unit ppm): ¹³ CNMR (126MHz, CDCl ₃ ) δ173.45, 172.41, 172.30, 150.34, 147.83, 144.26, 142.20, 141.10, 141.04 ,129.44,128.84,127.57,125.34,122.27,110.94,101.84,101.47,64.24,49.37,49.33,44.74,33.91,30.63,28.13,28.08,27.63,27.20,26.64,26.47,24.60,20.75,19.11,13.69.见Attached Figure 2.

Compound (Ⅲ) high resolution mass spectrum (HRMS) theoretical value C ₅₀ H ₆₈ ClN ₂ O ₄ [M] ⁺ 795.48621; experimental measurement: 795.49563. See attached drawing 3.

The H NMR signal of sensor RCy7 in deuterated chloroform is (500MHz, chemical shift, unit ppm): ¹ HNMR (500MHz, CDCl ₃ ) δ9.63(s, 1H), 7.91(d, J=5.9Hz, 1H ),7.64–7.52(m,4H),7.34–7.30(m,2H),7.27(s,1H),7.17(d,J=5.9Hz,1H),7.10(t,J=7.4Hz,2H) ,6.90(d,J=7.4Hz,2H),6.52(d,J=8.8Hz,2H),6.41(s,2H),6.34(d,J=8.8Hz,2H),5.63(s,1H) ,5.61(s,1H),4.08(t,J＝6.6Hz,4H),3.85(s,4H),3.48–3.54(m,4H),3.45–3.27(m,8H),2.50(s,4H ),2.35(t,J＝7.2Hz,4H),1.90–1.76(m,6H),1.75–1.69(m,6H),1.59–1.61(m,15H),1.49(d,J＝6.8Hz, 4H), 1.36–1.40 (m, 4H), 1.19 (t, J=6.9Hz, 12H), 0.94 (t, J=7.4Hz, 6H). See attached drawing 4.

In deuterated chloroform, the sensor RCy7 C NMR spectrum signal is (125MHz, chemical shift, unit ppm): ¹³ CNMR (125MHz, CDCl ₃ ) δ173.52, 170.43, 169.00, 166.91, 153.88, 153.46, 149.10, 143.09, 140.10, 137.12 ,133.45,129.95,128.56,128.29,124.28,122.81,122.65,121.78,119.39,108.58,108.39,104.11,98.01,94.19,64.29,52.43,47.51,44.46,43.09,42.12,34.03,31.94,30.66,29.71,29.37 , 28.63, 26.66, 26.31, 26.03, 24.67, 22.70, 21.11, 19.14, 14.13, 13.72, 12.65. See attached drawing 5.

Fluorescence sensor RCy7 high resolution mass spectrometry (HRMS) theoretical value C ₈₀ H ₁₀₃ N ₆ O ₆ [M] ⁺ 1243.79336; experimental measurement: 1243.84853. See attached drawing 6.

According to the analysis data, it can be judged that the substance is the fluorescence sensor RCy7 of the present invention.

Embodiment 2: The influence of the concentration of divalent copper ions on the ultraviolet-visible absorption spectrum of sensor RCy7 of the present invention

At a concentration of 10 μmol/Lol/L sensor RCy7 in acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) buffer solution, add 0,0.1,0.3,0.5,0.7,1,1.5,2.0, 3.0, 5.0, 5.0, 7.0, 10, 13, 15, 18, 20, 25, 30 times the equivalent concentration of divalent copper ion solution and measure the ultraviolet-visible absorption spectrum of the system, the results show that the maximum absorption value of the sensor RCy7 is at 633nm About, after adding divalent copper ions, the absorption at 633nm decreases significantly, and at the same time the absorption peak at 511nm continues to increase, but when the concentration of divalent copper ions is greater than 2 equivalents, the absorption peak at 511nm begins to decrease gradually . See attached drawing 7.

Embodiment 3: The fluorescent sensor RCy7 of the present invention can be used to detect divalent copper ions, and the specific application method is as follows:

Determination of divalent copper ion content: First, mix acetonitrile and Tris-HCl (pH=7.2) buffer evenly according to the volume ratio of 1:1, then add the sensor RCy7 of the present invention to make the concentration 10 μmol/L to make a standard solvent, Then use the prepared standard solvent to prepare divalent copper ion concentrations of 0, 1, 3, 5, 7, 10, 12, 13, 14, 15, 20, 30, 35, 40, 45, 50, 70, 100, 130, 150, 180, 200, 250, 300 μmol The copper ion standard solution of /L adopts HITACHI F2500 fluorescence spectrophotometer, and the excitation wavelength is 550nm, and the emission wavelength when measuring different copper ion concentrations is 577nm and 722nm when fluorescence intensity, fluorescence intensity ratio (F ₅₇₇ /F ₇₂₂ ) Make a standard curve with the concentration of copper ions, then add the fluorescent sensor of the present invention equally in the sample to be tested, so that its concentration is 10 μ mol/L, adopt the same method to measure the fluorescence intensity when the emission wavelength of the sample to be tested is 577nm and 722nm, and then Calculate the copper ion concentration in the sample to be tested according to the standard curve. See attached drawing 8.

Example 4: Determination of the selectivity of the fluorescent sensor RCy7 of the present invention to divalent copper ions by ultraviolet-visible absorption spectroscopy

The synthesized sensor RCy7 was prepared into 10 mM mother solution with dimethyl sulfoxide, and 50 mM various metal salt solutions were prepared with deionized water. Add 3 mL of acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) buffer solution to different cuvettes, and then add 3 μL of the mother solution of the sensor RCy7 of the present invention configured above to dilute it 10μmol/L. Then 3 μL of 50 mM various metal salt solutions were added thereto, and after mixing evenly, the ultraviolet-visible absorption spectrum of the system in the range of 300nm-800nm was measured. Sensor RCy7 has an efficient recognition performance for divalent copper ions: sensor RCy7 has a strong absorption peak at 633nm, and only after adding 3 μL of 50mM divalent copper ions, the strong emission peak at 633nm disappears, see Figure 9. This result indicates that the sensor RCy7 has a high UV-visible absorption selective recognition ability for divalent copper ions.

Example 5: Determination of the selectivity of the fluorescent sensor RCy7 of the present invention to divalent copper ions by fluorescence spectroscopy

The synthesized sensor RCy7 was prepared into 10 mM mother solution with dimethyl sulfoxide, and 50 mM various metal salt solutions were prepared with deionized water. Add 3 mL of acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) buffer solution to different cuvettes, and then add 3 μL of the mother solution of the sensor RCy7 of the present invention configured above to dilute it 10μmol/L. Then add 3μL of 50mM various metal salt solutions to it respectively, after mixing evenly, select the excitation wavelength as 550nm, measure the fluorescence spectrum of the system in the range of 560nm-800nm, the sensor RCy7 has efficient recognition performance for divalent copper ions: the sensor RCy7 has a strong emission peak at 722nm and a weak emission peak at 577nm. Only after adding 3 μL of 50mM divalent copper ions, the strong emission peak at 722nm disappears, and a strong emission peak appears at 577nm, see Figure 10. This result indicates that the sensor RCy7 also has a high fluorescence selective recognition ability for divalent copper ions.

Example 6: Effects of coexisting metal ions on the fluorescence detection of divalent copper ions by the sensor RCy7

In the acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) buffer solution with a concentration of 10 μmol/L sensor RCy7, after adding 50 μmol/L divalent copper ions, the fluorescence intensity ratio (F ₅₇₇ /F ₇₂₂ ) significantly increased. Then add other metal ions such as K ⁺ , Na ⁺ , Ca ²⁺ , Mg ²⁺ , Ba ²⁺ , Al ³⁺ , Mn ²⁺ , Cr ³⁺ , Cd ²⁺ , Pb ² into the sensor Cu ²⁺ solution respectively ⁺ ,Co ²⁺ ,Ag ⁺ ,Zn ²⁺ ,Fe ²⁺ ,Fe ³⁺ ,Hg ²⁺ ,Ni ²⁺ (concentration is equivalent to Cu ²⁺ ) after measuring the fluorescence intensity ratio of the system (F ₅₇₇ /F ₇₂₂ ) , the results showed that the presence of other metal ions did not affect the detection of divalent copper ions by the sensor RCy7 of the present invention. It reflects the strong anti-interference performance of the sensor RCy7. See attached drawing 11.

Example 7: Visualization of the selectivity of the fluorescent sensor RCy7 of the present invention to divalent copper ions

After adding 50 μmol/L of different metal ions to the acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) buffer solution with a concentration of 10 μmol/L sensor RCy7, the photos were taken under natural light. The results showed that the sensor RCy7 changed from blue to light pink only after the addition of divalent copper ions. This intuitively indicates that the sensor RCy7 is highly selective for divalent copper ions. See attached drawing 12.

Embodiment 8: The fluorescent sensor RCy7 of the present invention can be used to detect pH, and the specific application method is as follows:

pH measurement: first mix acetonitrile and water evenly according to the volume ratio of 1:1, then add the sensor RCy7 of the present invention to make the concentration 10 μmol/L to make a standard solvent, and then use NaOH solution and hydrochloric acid to make pH 1.26, 1.29 , 1.32, 1.35, 1.39, 1.47, 1.58, 1.77, 2.01, 2.24, 2.43, 2.64, 3.01, 3.37, 3.61, 3.74, 3.91, 4.06, 5.46, 6.58 solutions, using HITACHI F2500 fluorescence spectrophotometer, at the excitation wavelength Fluorescence intensity at 580nm and 722nm when the emission wavelength is measured at different pHs respectively, and the fluorescence intensity ratio (F ₅₈₀ /F ₇₂₂ ) and pH make a standard curve, and then add the fluorescence sensor of the present invention to the sample to be tested to make it The concentration is 10 μmol/L, and the fluorescence intensity of the sample to be tested is measured by the same method when the emission wavelength is 580nm and 722nm, and then the pH of the sample to be tested is calculated according to the standard curve. See attached drawing 13. It can be seen from the figure that the fluorescence sensor RCy7 of the present invention can detect the pH of the strong acid system with high near-infrared ratio and selectivity, and the pH range of the strong acid system is 1.26-2.43.

Example 9: Selectivity of the fluorescent sensor RCy7 of the present invention to pH

The synthesized sensor RCy7 was prepared into 10 mM mother solution with dimethyl sulfoxide, and 50 mM various metal salt solutions were prepared with deionized water. Add 3 mL of acetonitrile/Tris-HCl (v/v, 1:1, pH=7.2) buffer solution to different cuvettes, and then add 3 μL of the mother solution of the sensor RCy7 of the present invention configured above to dilute it 10μmol/L. Then add 3μL of 50mM various metal salt solutions to it respectively, after mixing evenly, select the excitation wavelength as 550nm, measure the fluorescence spectrum of the system in the range of 560nm-800nm, the sensor RCy7 has efficient recognition performance for divalent copper ions: the sensor RCy7 has a strong emission peak at 722nm and a weak emission peak at 577nm. Only after adding 3 μL of 50mM divalent copper ions, the strong emission peak at 722nm disappears, and a strong emission peak appears at 577nm, see Figure 10. However, when the pH of the solution is less than 2.64, the strong emission peak at 722nm gradually disappears, and the strong emission peak at 580nm is greatly enhanced, and the fluorescence signal changes caused by other metal ions are relatively small, and the sensor RCy7 has a high degree of fluorescence selectivity for pH sexual recognition. See attached drawing 14.

Example 10: Laser confocal fluorescence microscopy imaging of exogenous divalent copper ions in cells by fluorescence sensor RCy7 of the present invention

We applied the fluorescent sensor RCy7 of the present invention to the laser confocal fluorescence microscopy imaging application of exogenous divalent copper ions in Hela living cells. The specific operation steps are as follows: 10 μmol/L fluorescent sensor RCy7 was added to the culture medium with Hela cells at 37°C for 60 min, and then fluorescent imaging was performed with a confocal microscope. First perform bright field imaging, you can see the rough outline of the cells, and then use 552nm light to excite and observe the fluorescence imaging situation before adding Cu ²⁺ , at this time, the fluorescence emission of the observation channel (560nm-650nm) is very weak, and the channel (680nm-800nm) has strong fluorescence. After adding 50 μmol/L Cu ²⁺ aqueous solution to the system, wait 60 minutes and then excite with 552nm light, it can be observed that the fluorescence emission of the channel (560nm-650nm) is enhanced, and the fluorescence of the channel (650nm-800nm) disappears. It shows that the fluorescence sensor RCy7 can perform fluorescence imaging of exogenous divalent copper ions. The specific results are shown in Figure 15.

Example 11: Laser confocal fluorescence microscopy imaging of Escherichia coli bacteria in different pH systems of the fluorescent sensor RCy7 of the present invention

We applied the fluorescent sensor RCy7 of the present invention to the application of laser confocal fluorescence microscopy imaging of protons in E.coli live bacteria. The specific operation steps are as follows: E. coli was placed in LB medium at 37° C. on a shaker at 180 rpm for 18 hours. Then take out 10mL, centrifuge at 5000rpm for 5min to remove the culture medium, wash twice with sterile water, divide the precipitate into two tubes, and add aqueous hydrochloric acid solutions with pH=7.4 and pH=1.8 respectively, and after 5min each Add probe RCy7 to each tube to make the final probe concentration 10 μmol/L, then place on a shaker at 37°C and 180 rpm for 2 hours, and centrifuge at 5000 rpm for 5 minutes to remove the probe solution, wash twice with PBS, and coat the bacteria On glass slides, confocal imaging of bacteria was performed (LeicaTCS SP8 laser confocal microscope). The excitation wavelength of the laser is 552nm, and the emission wavelength collection channels are 560nm-650nm and 680nm-800nm. When pH=7.4, the fluorescence emission of the observation channel (560nm-650nm) is very weak, and the fluorescence emission of the channel (680nm-800nm) is strong. When pH=1.8, the fluorescence emission of the channel (560nm-650nm) can be observed to increase and the fluorescence of the channel (650nm-800nm) to disappear when excited with 552nm light. It shows that the fluorescence sensor RCy7 can perform fluorescence imaging on the pH of E.coli living bacteria in strong acid system. The specific results are shown in Figure 16.

In the coexistence system of divalent copper ions and other various metal ions, the fluorescence sensor RCy7 of the present invention exhibits high sensitivity, good selectivity, insensitive to pH, and can realize near-infrared ratio detection of divalent copper ions; and can realize near-infrared High ratio selectivity for pH detection of strong acid systems. It can be applied to fluorescence microscopy imaging of divalent copper ions in biological living cells. It can be applied to pH fluorescence imaging of E.coli living bacteria in strong acid system.

The above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present invention. Should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (9)

1. A class of compounds, characterized in that, have the following general structural formula I: In general formula I: a is 1-6. 2. The preparation method of a class of compounds described in claim 1, characterized in that, in N,N-dimethylformamide solution, the obtained compound II reacts with the general structural formula III, 3. A preparation method of a compound, characterized in that 2,3,3-trimethyl-3H-indoline is dissolved in acetonitrile and reacted with 6-bromohexanoic acid to generate compound IV, compound IV and condensing agent 2-Chloro-1-formyl-3-hydroxymethylcyclohexene was reacted to generate compound III; rhodamine B was dissolved in ethanol and reacted with ethylenediamine to generate compound II; finally compound III and compound II were dissolved in N, Compound I is obtained after reacting in N-dimethylformamide; The compound IV is: The compound III is: The compound II is: The compound I is: 4. the preparation method of a kind of compound according to claim 3, is characterized in that, the molar ratio of described 2,3,3-trimethyl-3H-indoline and 6-bromohexanoic acid is 1: 1-3. 5. the preparation method of a kind of compound according to claim 3 is characterized in that, the molar ratio of the condensing agent 2-chloro-1-formyl-3-hydroxymethylcyclohexene and compound IV is 1 :2.1-5. 6. the preparation method of a kind of compound according to claim 3, is characterized in that, the molar ratio of described rhodamine B and ethylenediamine is 1:1-5. 7. The preparation method of a compound according to claim 3, characterized in that, the molar ratio of compound III to compound II is 1:1-5. 8. The application of a class of compounds as claimed in claim 1 in detecting pH value as a ratiometric fluorescent sensor. 9. The application of a class of compounds described in claim 1 as a ratiometric fluorescent sensor in the detection of divalent copper ions.